From stems cells to neurons, precise gene regulation is required for growth and prevents the formation of disease. Due to intrinsic copy number variation throughout the genome 1, coordinate regulation of a large number of genes is essential. Recently, coordinate gene regulation of sub-nuclear domains were found to be nucleated by long non-coding RNAs throughout the human genome but their establishment remains poorly understood 2. The X-chromosome in Drosophila males is an important model for the establishment of these domains because all of the genes on a single chromosome are specifically upregulated. This process of dosage compensation involves many highly-conserved proteins and several well-defined non-coding RNAs which together increases transcript levels of the single X chromosome in males by two-fold to equalize expression levels with the two X-chromosomes in females and with the other chromosomes 3. The key regulator of dosage compensation in Drosophila is the Male Specific Lethal (MSL) ribonucleoprotein complex, which is specifically ex- pressed in males and distinguishes the X-chromosome from the autosomes. The overall objective of this proposal is to define the critical first steps in X-identification. It is known that the MSL complex is assembled during the transcription of its two non-coding RNA components, RNA on X (roX), which are encoded on the X- chromosome 4,5 and act as "seed" sites for MSL complex recruitment. These "seed" sites also contain MSL Recognition Elements (MREs), which are key 21 bp cis-acting sequences that are two-fold X-enriched. The MRE sequences are also required for MSL complex recruitment 6. However, it is not known how the MREs and non-coding RNAs are functionally integrated to generate X-specificity because the MREs are not X-specific and the MSL components are not sufficient to target the MREs 7. Using an innovative screening approach, we recently identified an essential zinc-finger protein that is strongly enriched at the MREs. Surprisingly, the MSL complex and the zinc-finger protein associate inter-dependently at MSL complex high affinity sites distributed across the X-chromosome. Guided by strong preliminary data, our central hypothesis is that the MSL complex recognizes the X-chromosome in males by integrating the functions of non-coding RNAs and cis-acting sequences to generate a novel positive feedback mechanism, which amplifies the two-fold enrichment of the MRE sequences to generate X-specificity. To rigorously test our hypothesis, we will use our new cell-based MSL complex induction system to define the dynamics and requirements for MSL complex recruitment to the RoX "seed" sites in real-time (Aim #1). Furthermore, we will determine how MSL complex is recruited to cis-acting sequences distant from the roX loci by retargeting the zinc-finger protein to a new locus (Aim #2). Our innovative model is likely to define a new paradigm for sub-nuclear domain formation across species because it is the first mechanism that integrates the functions of non-coding RNAs and cis-acting sequences. We expect that defining how genes are coordinately regulated will provide key therapeutic targets for diverse diseases. PUBLIC HEALTH RELEVANCE: The overall objective of this proposal is to define the critical first step in X-identification during dosage compensation in Drosophila. Elucidating how genes are targeted for coordinate regulation is relevant to the part of the NIH mission that aims to provide the critical foundation of knowledge for future disease therapies. Specifically, the proposed research will guide the development of new therapeutic approaches for diseases that are associated with loss of coordinate regulation such as several cancers 8, autism 9, Crohn's disease 10, and Down syndrome 11. !